Plant plastid transformation method

Abstract

Method for heterologous protein production in plant cell plastids comprising introducing into plant cells nucleic acid components that encode heterologous proteins under the control of promoters operative in plastids, vectors, host cells, plants and uses thereof.

Claims

1. A method of transforming the plastids in a tobacco or Arabidopsis plant cell, the method comprising: 1) introducing into the nucleus of the plant cell a first nucleic acid construct comprising a Lactococcus lactis LtrB, an expression cassette comprising a left flanking sequence, a plastid specific promoter, a nucleic acid of interest, a plastid specific terminator, and a right flanking sequence, and a nucleic acid comprising a primer binding domain (PBD) from tobacco tnt1 retrotransposon or yeast Ty1 retrotransposon; and 2) introducing into the nucleus of the plant cell a second nucleic acid construct encoding a first transit peptide operably linked to a Lactococcus lactis LtrA protein, wherein the left and right flanking sequences initiate homologous recombination of the expression cassette into a plastid genome; wherein said first and second nucleic acid constructs are operably linked to a plant nuclear promoter; and wherein the first and second transit peptides are a small subunit of Rubisco or HSP70.

2. A method of transforming the plastids in a tobacco or Arabidopsis plant cell, the method comprising: 1) introducing into the nucleus of the plant cell a first nucleic acid sequence comprising a DNA sequence coding for avocado sunblotch viroid (ASB) RNA, an expression cassette comprising a left flanking sequence, a plastid specific promoter, a nucleic acid of interest, a plastid specific terminator, and a right flanking sequence, and a nucleic acid comprising a primer binding domain (PBD) from tobacco tnt1 retrotransposon or yeast Ty1 retrotransposon; and 2) introducing into the nucleus of the plant cell a second nucleic acid sequence encoding a transit peptide, wherein said transit peptide is from a small subunit of Rubisco or HSP70, operably linked to a reverse transcriptase protein (RTP), wherein said reverse transcriptase protein is selected from the group consisting of retrotransposon encoded reverse transcriptase and retroviral reverse transcriptase, wherein said first and second nucleic acid sequences are operably linked to a plant nuclear promoter.

3. The method according to claim 2, wherein the reverse transcriptase is encoded by a nucleic acid sequence from a yeast retrotransposon Ty1.

4. A method of producing at least a heterologous or exogenous RNA species in a tobacco or Arabidopsis plant, the method comprising: (1) introducing into a regenerable plant cell a first nucleic acid construct comprising a Lactococcus lactis LtrB, an expression cassette comprising a left flanking sequence, a plastid specific promoter, a nucleic acid of interest, a plastid specific terminator, and a right flanking sequence, and a nucleic acid comprising a primer binding domain (PBD) from a tobacco tnt1 retrotransposon or a yeast Ty1 retrotransposon; and (2) introducing into the regenerable plant cell a second nucleic acid construct encoding a first transit peptide operably linked a Lactococcus lactis LtrA protein (a) wherein said first and second nucleic acid constructs are operably linked to a plant nuclear promoter; and (b) wherein the first and second transit peptides are from a small subunit of Rubisco or HSP70 (3) growing a regenerable plant cell comprising the first and second nucleic acid constructs; (4) selecting a plant cell of step (3), wherein the expression construct is integrated into the plastid genome; (5) regenerating a plant from the plant cell of step (4); and (6) growing the plant of step (5), wherein the expression construct that is integrated into the plastid expresses the nucleic acid of interest encoding a heterologous or exogenous protein.

5. A method of producing at least a heterologous or exogenous RNA species in a tobacco or Arabidopsis plant, the method comprising: (1) introducing into a regenerable plant cell a first nucleic acid construct comprising a DNA sequence coding for avocado sunblotch viroid (ASB) RNA, an expression cassette comprising a left flanking sequence, a plastid specific promoter, a nucleic acid of interest, a plastid specific terminator, and a right flanking sequence, and a nucleic acid comprising a primer binding domain (PBD) from tobacco tnt1 retrotransposon or yeast Ty1 retrotransposon; (2) introducing into the regenerable plant cell a second nucleic acid construct encoding a transit peptide, said transit peptide from a small subunit of Rubisco or HSP70, operably linked to a reverse transcriptase protein (RTP), wherein said reverse transcriptase protein is selected from the group consisting of retrotransposon encoded reverse transcriptase and retroviral reverse transcriptase; wherein said first and second nucleic acid sequences are operably linked to a plant nuclear promoter; (3) growing a regenerable plant cell comprising the first and second nucleic acid constructs; (4) selecting a plant cell of step (3), wherein the expression construct is integrated into the plastid genome; (5) regenerating a plant from the plant cell of step (4); and (6) growing the plant of step (5), wherein the expression construct that is integrated into the plastid expresses the nucleic acid of interest encoding a heterologous or exogenous protein.

6. A polynucleotide construct that comprises a plant nuclear promoter operably linked to a first nucleic acid sequence comprising a plant plastid translocation sequence wherein said plastid translocation sequence is selected from the group consisting of Lactococcus lactis LtrB or avocado sunblotch viroid RNA; an expression cassette comprising a left flanking sequence, a plastid specific promoter, a nucleic acid of interest, a plastid specific terminator, and a right flanking sequence; and a primer binding domain (PBD) from tobacco tnt1 retrotransposon or yeast Ty1 retrotransposon.

7. A polynucleotide construct that comprises a plant nuclear promoter operably linked to a nucleic acid sequence encoding a plant plastid transit peptide operably linked to a Lactococcus lactis LtrA; wherein said plant plastid transit peptide is from a small subunit of Rubisco or HSP70 proteins.

8. A polynucleotide construct that comprises a plant nuclear promoter operably linked to a nucleic acid sequence encoding a plant plastid transit peptide operably linked to a reverse transcriptase protein, wherein said reverse transcriptase protein is selected from the group consisting of retrotransposon encoded reverse transcriptase and retroviral reverse transcriptase; wherein said plant plastid transit peptide is from a small subunit of Rubisco or HSP70 proteins; and wherein said plant plastid transit peptide is heterologous with respect to said retroviral reverse transcriptase.

9. A plant comprising the construct according to claim 6.

10. A method of producing a plant, the method comprising incorporating the construct according to claim 9 into an Arabidopsis or tobacco plant cell and regenerating a plant from said plant cell.

11. The method according to claim 2, wherein the nucleic acid construct encoding said reverse transcriptase protein is from yeast retrotransposon Ty1 or RNase H.

Description

FIGURES

(1) FIG. 1: the major components of chloroplast transformation system.

(2) (1) Transformation vector contains (i) chloroplast translocation sequence (CTS); (ii) chloroplast transgene cassette comprising left flanking sequence (LFS) and right flanking sequence (RFS) to facilitate insertion of the cassette into the chloroplast genome using homologous recombination, promoter region from tobacco chloroplast rrn16 gene (Prrn), aadA gene as a selectable marker (aadA), transcription terminator from chloroplast genome (term); and (iii) primer binding domain (PBD). (2) Reverse Transcriptase-RNase H gene translationally fused to the chloroplast transit peptide from small subunit of tobacco Rubisco gene (rbcS-cTP). (3) CTS-Binding peptide translationally fused to the chloroplast transit peptide from Arabidopsis HSP60 gene (HSP60-cTP).

(3) FIG. 2: set of constructs used for chloroplast transformation in tobacco (ALG298, ALG327 and ALG 344) and in Arabidopsis (ALG347 and ALG327)

(4) The chloroplast transformation cassette contains left and right flanking sequences (LFS and RFS), Prrn promoter (Prrn), aadA gene for spectinomycin selection (aadA), and rrnB transcription terminator (rrnB ter). Primer binding domain (PBD) from yeast Ty1 retrotransposon designed for capturing tRNA-Met from chloroplasts was fused to chloroplast transgene cassette. The resulting cassette was inserted within domain IV of LtrB (LtrB5 and LtrB3) intron from Lactococcus lactis (ALG298 and ALG347) or fused to chloroplast translocation sequence from Avocado sunblotch viroid (ASB-CTS in ALG344). The chloroplast transgene cassette was expressed from nuclear inserted cassette and resultant RNA was translocated into the chloroplast using LtrASi protein for vectors (ALG298 and ALG347), or using native plant proteins for vector ALG344. Reverse transcription of the RNA was performed by Reverse transcriptase-RNaseH fused to chloroplast transit peptide (cTPRTRH) from HSP60 gene (ALG327). Ubiq3 ProArabidopsis promoter from ubiquitin 3 gene; 35S Propromoter from Cauliflower Mosaic Virus 35S gene, TAF2 ProArabidopsis promoter from TAF 2 gene; nos tertranscription terminator from Agrobacterium nos gene.

(5) FIG. 3: modifications of the chloroplast transformation cassette were made by designing primer binding domain and positioning of building blocks on the transgene cassette.

(6) CTUchloroplast transformation unit; CTS-5-chloroplast translocation sequence located at the 5-end of the transformation cassette; CTS-3chloroplast translocation sequence located at the 3-end of the transformation cassette; PDB-CHLprimer binding domain designed for reverse transcription in the chloroplasts using tRNA-Met from chloroplasts; PBD-CYTprimer binding domain designed for reverse transcription in the cytoplasm using cytoplasmic tRNA-Met.

(7) The modifications detailed in Example section 1B hereinafter and corresponding figures include a first modification of the use of PBD for the binding of cytoplasmic tRNA-Met as primer [FIG. 3(C)]. As a second modification CTS can be located at both the 5- and 3-ends of the transformation cassette, such as in the case with the LtrB intron. The transgene cassette is inserted inside of the LtrB intron (domain IV). The PDB-CHL is located downstream of the LtrB 3-end of the cassette (CTS-3), so that the LtrA protein is able to function as both a translocation protein and reverse transcriptase. The LtrA protein has three major functions: (1) as a maturase (it binds to LtrB RNA and stabilises the secondary structure of the RNA, and assists splicing); (2) as an endonuclease (it induces single-stranded DNA breaks on target site); and (3) as a reverse transcriptase (it performs reverse transcription of the intron RNA after insertion of the LtrB intron RNA into the donor site).

(8) The LtrA protein is unable to perform the reverse transcription reaction efficiently if the PBD-CYT is located adjacent to and in front of a chloroplast translocation sequence at the 3-end of the CTU (CTS-3) as in FIG. 3(B), but can efficiently reverse transcribe RNA if the PBD is located downstream of a chloroplast translocation sequence (CTS-3) as shown in FIG. 3A. Such a positioning or the combination of components of the transformation cassette as shown in FIG. 3(A) allows both the translocation of the CTU into the chloroplast and reverse transcription of the CTU by the LtrA protein. Thus, by positioning of the CTS components and of the PBD-CHL as shown in FIG. 3(A) the procedure of transformation is simplified since there is no requirement to co-deliver another gene to provide a reverse transcriptase function.

(9) A similar simplification of the procedure is achieved if a PBD-CYT is used, since there is a significant amount of native endogenous reverse transcriptase in the cytoplasm, and reverse transcription is initiated by endogenous reverse transcriptase using cytoplasmic tRNA-Met. This also eliminates the necessity for the co-delivery of another gene for reverse transcription in the chloroplasts.

(10) The case in FIGS. 1A and B is attributed to LtrB intron, the case in FIG. 1C attributed to ASB-CTS.

(11) FIG. 4: schematic presentation of constructs based on the LtrB-CTS for chloroplast transformation in tobacco.

(12) Nos ternos transcription terminator, LtrB33-prime end of LtrB intron, PBD-CHLprimer binding domain for chloroplast tRNA-Met, PDB-CYTprimer binding domain for cytoplasmic tRNA-Met, trnA flankleft flank of the transgene cassette, psbA terchloroplast transcription terminator from tobacco, mGFPmGFP4 gene, aadA-aadA gene, Trrnrrn16 chloroplast promoter from tobacco, trnI flankright flank of transgene cassette, LtrB55-prime end of LtrB intron, 35S Pro35S promoter from cauliflower mosaic virus (CaMV), TAF2 Propromoter from Arabidopsis TAF2 gene, cTPchloroplast transit peptide from rbcS gene of tobacco, LtrAgene encoded by open reading frame of LtrB intron, ags terags gene transcription terminator.

(13) FIG. 5: schematic presentation of constructs based on the LtrB-CTS for chloroplast transformation in rice.

(14) Nos ternos transcription terminator, LtrB33-prime end of LtrB intron, PBD-CHLprimer binding domain for chloroplast tRNA-Met, PDB-CYTprimer binding domain for cytoplasmic tRNA-Met, trnA flankleft flank of the transgene cassette, atpA terchloroplast transcription terminator from wheat, mGFPmGFP4 gene, aadAaadA gene, Wrrnrrn16 chloroplast promoter from wheat, trnI flank-right flank of transgene cassette, LtrB55-prime end of LtrB intron, 35S Pro35S promoter from cauliflower mosaic virus (CaMV), Act1 Proactin 1 gene promoter from rice, cTPchloroplast transit peptide from rbcS gene of tobacco, LtrAgene encoded by open reading frame of LtrB intron, ags terags gene transcription terminator.

(15) FIG. 6: Schematic presentation of constructs based on ASB-CTS for chloroplast transformation in tobacco.

(16) Nos ternos transcription terminator, ASBsequence from Avocado sunblotch viroid (ASBVd) as CTS, PBD-CHLprimer binding domain for chloroplast tRNA-Met, PDB-CYTprimer binding domain for cytoplasmic tRNA-Met, trnA flankleft flank of the transgene cassette, psbA terchloroplast transcription terminator from tobacco, mGFPmGFP4 gene, aadAaadA gene, Trrnrrn16 chloroplast promoter from tobacco, trnI flankright flank of transgene cassette, 35S Pro35S promoter from cauliflower mosaic virus (CaMV), TAF2 Propromoter from Arabidopsis TAF2 gene, cTPchloroplast transit peptide from rbcS gene of tobacco, RT-ty1reverse transcriptase gene from yeast ty1 retrotransposon, ags terags gene transcription terminator.

(17) FIG. 7: PCR amplification of left flanking junction in tobacco transformed by the LtrB-CTS-based vectors.

(18) MDNA marker, 1-6independent transgenic lines, wtnon-transgenic tobacco, NCnegative control without DNA.

(19) FIG. 8: Southern hybridisation for tobacco transformed with ASB-CTS and LtrB-CTS based vectors.

(20) Expected size of wild type DNA band is 1.3 kb, and band with transgene insertion 3.6 kb. Chloroplast probe upstream of LFS was used as a probe. MDNA marker, wtDNA from non-transgenic line, 1-3ASB-CTS lines, 4-8LtrB-CTS transgenic lines.

(21) FIG. 9: Northern analysis for tobacco plants transformed with LtrB-CTS based vector.

(22) The aad-GFP DNA probe was used for hybridisation. Expected size of the band is 1.5 kb. Lane 1RNA from plants transformed with 35S-aadA-GFP-nos cassette; lane 2WT RNA; lanes 3-8independent transgenic lines.

EXPERIMENTAL SECTION 1A

A Novel Approach for Efficient Chloroplast Transformation

(23) A new method for chloroplast transformation in plants comprises (1) a transformation vector consisting of 3 major domains: (i) chloroplast translocation sequence (CTS), (ii) chloroplast transgene cassette, (iii) primer binding domain (PBD) which uses chloroplast tRNA-fMet or any other chloroplast tRNAs as a primer for reverse transcription;

(24) (2) Reverse TranscriptaseRNase H (RT-RH) from retrotransposon or retroviruses fused to chloroplast transit peptide for targeting into chloroplasts;

(25) (3) RNA binding protein that binds to chloroplast translocation sequence (CTS) of the transformation vector, fused to chloroplast transit peptide (FIG. 1).

(26) Technology Rationale

(27) The process of chloroplast transformation comprises two steps:

(28) (1) Targeting of RNA-Protein Complex to the Chloroplasts.

(29) After delivery of the chloroplast transformation construct into the plant cell a strong expression of the RNA which contains the chloroplast translocation sequence (CTS) transgene cassette and primer binding domain (PBD) is achieved from the nuclear specific promoter. The CTS binding protein (CTS-BP) fused to a chloroplast transit peptide, will be also over-expressed on co-delivery from the same or a different vector and then will bind to the CTS, and facilitate translocation of the RNA into the chloroplasts.

(30) Once the chloroplast transformation vector is presented in the plant cell via nuclear transformation, the chloroplast will then be permanently bombarded by the expressed CTS-BP-RNA complex. Such stable and continuous pumping of the complex into the targeted organelle is a prerequisite for achieving a high efficiency of organelle transformation. The technology exploits the finding that the chloroplast transit sequence is sufficient to permit the whole CTS-BP-RNA complex to be then taken up by the chloroplast.

(31) Chloroplast translocation sequence (CTS) can be selected from a number of RNA sequences such as viroid RNA, groupI and groupII intron RNA, viral coat protein binding domains, retrotransposon primer binding sites, which are recognised by corresponding native RNA binding proteins.

(32) (2) Reverse Transcription of the Transgene Cassette and Insertion into the Chloroplast Genome.

(33) Once the RNA of the transformation vector is inside of the organelle, primer binding domain (PBD) of the vector RNA captures tRNA-fMet as a primer, and the over-expression of the reverse transcriptase (RT-RH) fused to the chloroplast transit peptide facilitates reverse transcription of RNA into single stranded DNA. This is followed by insertion of the reverse transcribed cassette into the chloroplast genome using homologous recombination between flanking sequences of the transgene cassette and the homologous regions in the chloroplast genome.

(34) Primer binding domain (PBD) is designed to capture RT-RH protein and chloroplast tRNA-fMet (or other chloroplast tRNAs) as a primer, and initiate reverse transcription of chloroplast transgene cassette RNA into single-stranded DNA.

(35) Once the population of organelle genomes has been transformed in the initial plant line, the nuclear encoded transgenes are no longer required and they can then be removed through segregation in subsequent plant generations, leaving a clean organelle transformed plant line.

(36) Materials and Methods

Preparation of Group II Intron Based Chloroplast Translocation Sequence (CTS)

(37) LtrB intron from Lactococcus lactis was synthesised by commercial DNA synthesis provider. Potential splicing sites were eliminated from this sequence as described in our previous patent. The domain for insertion of transgene cassette (AscI-MluI-NotI sites) is underlined and shown in bold letters.

(38) TABLE-US-00001 LtrBintronsequence SEQIDNO.1 GGATCCCTCGAGGTGCGCCCAGATAGGGTGTTAAGTCAAGTAGTTTAAGGTACTACTCAGTAAGAT AACACTGAAAACAGCCAACCTAACCGAAAAGCGAAAGCTGATACGGGAACAGAGCACGGTTGGAAA GCGATGAGTTAGCTAAAGACAATCGGCTACGACTGAGTCGCAATGTTAATCAGATATAAGCTATAA GTTGTGTTTACTGAACGCAAGTTTCTAATTTCGGTTATGTGTCGATAGAGGAAAGTGTCTGAAACC TCTAGTACAAAGAAAGCTAAGTTATGGTTGTGGACTTAGCTGTTATCACCACATTTGTACAATCTG TTGGAGAACCAATGGGAACGAAACGAAAGCGATGGCGAGAATCTGAATTTACCAAGACTTAACACT AACTGGGGATAGCCTAAACAAGAATGCCTAATAGAAAGGAGGAAAAAGGCTATAGCACTAGAGCTT GAAAATCTTGCAAGGCTACGGAGTAGTCGTAGTAGTCTGAGAAGGCTAACGGCCTTTACATGGCAA AGGGCTACAGTTATTGTGTACTAAAATTAAAAATTGATTAGGGAGGAAAACCTCAAAATGAAACCA ACAATGGCAATTTTAGAAAGAATCAGTAAAAATTCACAAGAAAATATAGACGAAGTTTTTACAAGA CTTTATCGTTATCTTTTACGTCCTGATATTTATTACGTGGCGGGCGCGCCACGCGTGCGGCCGCTG GGAAATGGCAATGATAGCGAAAGAACCTAAAACTCTGGTTCTATGCTTTCATTGTCATCGTCACGT GATTCATAAACACAAGTGAATTTTTACGAACGAACAATAACAGAGCCGTATACTCCGAGAGGGGTA CGTACGGTTCCCGAAGAGGGTGGTGCAAACCAGTCACAGTAATGTGAACAAGGCGGTACCTCCCTA CTTCACCATATCATTTTTAATTCTACGAATCTTTATACTGGCAAACAATTTGACTG

(39) The chloroplast translocation sequence (CTL) from Avocado sunblotch viroid (Bank Accession No. J02020) was synthesised by PCR using the set of the following overlapping primers:

(40) TABLE-US-00002 AS839 SEQIDNO.2 GAACTAATTTTTTTAATAAAAGTTCACCACGACTCCTCCTTCTCTCACAA AS840 SEQIDNO.3 TAAAAAAATTAGTTCACTCGTCTTCAATCTCTTGATCACTTCGTCTCTTC AS841 SEQIDNO.4 TGCGAGACTCATCAGTGTTCTTCCCATCTTTCCCTGAAGAGACGAAGTGA AS842 SEQIDNO.5 CTGATGAGTCTCGCAAGGTTTACTCCTCTATCTTCATTGTTTTTTTACAA AS843 SEQIDNO.6 GGGCGCGCCAAGATTTTGTAAAAAAACAATGAAGA AS844 SEQIDNO.7 GCTCGAGACTTGTGAGAGAAGGAGGAGTC TheCTLsequencefromAvocadosunblotchviroid SEQIDNO.8 GCTCGAGACTTGTGAGAGAAGGAGGAGTCGTGGTGAACTTTTATTAAAAAAATTAGTTCACTCGTC TTCAATCTCTTGATCACTTCGTCTCTTCAGGGAAAGATGGGAAGAACACTGATGAGTCTCGCAAGG TTTACTCCTCTATCTTCATTGTTTTTTTACAAAATCTTGGGCGCGCCC

(41) The expression of chloroplast translocation sequence and chloroplast cassette fused to it was driven by 35S promoter from Cauliflower mosaic virus obtained by DNA synthesis

(42) TABLE-US-00003 35Spromotersequence SEQIDNO.9 CAATCCCACAAAAATCTGAGCTTAACAGCACAGTTGCTCCTCTCAGAGCAGAATCGGGTATTCAAC ACCCTCATATCAACTACTACGTTGTGTATAACGGTCCACATGCCGGTATATACGATGACTGGGGTT GTACAAAGGCGGCAACAAACGGCGTTCCCGGAGTTGCACACAAGAAATTTGCCACTATTACAGAGG CAAGAGCAGCAGCTGACGCGTACACAACAAGICAGCAAACAGACAGGTTGAACTTCATCCCCAAAG GAGAAGCTCAACTCAAGCCCAAGAGCTTTGCTAAGGCCCTAACAAGCCCACCAAAGCAAAAAGCCC ACTGGCTCACGCTAGGAACCAAAAGGCCCAGCAGTGATCCAGCCCCAAAAGAGATCTCCTTTGCCC CGGAGATTACAATGGACGATTTCCTCTATCTTTACGATCTAGGAAGGAAGTTCGAAGGTGAAGTAG ACGACACTATGTTCACCACTGATAATGAGAAGGTTAGCCTCTTCAATTTCAGAAAGAATGCTGACC CACAGATGGTTAGAGAGGCCTACGCAGCAGGTCTCATCAAGACGATCTACCCGAGTAACAATCTCC AGGAGATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAAGATTCAGGACTAATTGCATCA AGAACACAGAGAAAGACATATTTCTCAAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCT TGCTTCATAAACCAAGGCAAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCTACTGAATCTA AGGCCATGCATGGAGTCTAAGATTCAAATCGAGGATCTAACAGAACTCGCCGTGAAGACTGGCGAA CAGTTCATACAGAGTCTTTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCAC GACACTCTGGTCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACT TTTCAACAAAGGATAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATC GAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATC ATTCAAGATCTCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAA AAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGG GATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTG GAGAGGACACG

(43) The chloroplast transgene cassette contains left and right flanking sequences (LFS and RFS) for insertion of whole cassette into the chloroplast genome using homologous recombination, Prrn16 promoter region from tobacco, aadA gene as a selectable marker, and 3UTR sequence of psbA gene as transcription terminator (FIG. 1).

(44) LFS sequences for tobacco and Arabidopsis were amplified using the following PCR primers:

(45) TABLE-US-00004 AS699 SEQIDNO.10 GGCGCGCCGTGGGATCCGGGCGGTCCG AS700 SEQIDNO.11 GGCATGCTGGCGCAGCTGGGCCATCC TobaccoLFSsequence SEQIDNO.12 GGCGCGCCATGGGATCCGGGCGGTCCGGGGGGGACCACCACGGCTCCTCT CTTCTCGAGAATCCATACATCCCTTATCAGTGTATGGACAGCTATCTCTC GAGCACAGGTTTAGCAATGGGAAAATAAAATGGAGCACCTAACAACGCAT CTTCACAGACCAAGAACTACGAGATCGCCCCTTTCATTCTGGGGTGACGG AGGGATCGTACCATTCGAGCCGTTTTTTTCTTGACTCGAAATGGGAGCAG GTTTGAAAAAGGATCTTAGAGTGTCTAGGGTTGGGCCAGGAGGGTCTCTT AACGCCTTCTTTTTTCTTCTCATCGGAGTTATTTCACAAAGACTTGCCAG GGTAAGGAAGAAGGGGGGAACAAGCACACTTGGAGAGCGCAGTACAACGG AGAGTTGTATGCTGCGTTCGGGAAGGATGAATCGCTCCCGAAAAGGAATC TATTGATTCTCTCCCAATTGGTTGGACCGTAGGTGCGATGATTTACTTCA CGGGCGAGGTCTCTGGTTCAAGTCCAGGATGGCCGCATGCC ArabidopsisLFSsequence SEQIDNO.13 GGCGCGCCGTGGGATCCGGGCGGTCCGGAGGGGACCACTATGGCTCCTCT CTTCTCGAGAATCCATACATCCCTTATCAGTGTATGGACAGCTATCTCTC GAGCGCAGGTTTAGGTTCGGCCTCAATGGGAAAATAAAATGGAGCACCTA ACAACGTATCTTCACAGACCAAGAACTACGAGATCACCCCTTTCATTCTG GGGTGACGGAGGGATCGTACCGTTCGAGCCTTTTTTTCATGTTATCTATC TCTTGACTCGAAATGGGAGCAGGTTTGAAAAAGGATCTTAGAGTGTCTAG GGTTAGGCCAGTAGGGTCTCTTAACGCCCTCTTTTTTCTTCTCATCGAAG TTATTTCACAAATACTTCCTATGGTAACGAAGAGGGGGGGAACAAGCACA CTTGGAGAGCGCAGTACAACGGAGAGTTGTATGCTGCGTTCGGGAAGGAT GAATCGCTCCCGAAAAGGAATCTATTGATTCTCTCCCAATTGGTTGGACC ATAGGTGCGATGATTTACTTCACGGGCGAGGTCTCTGGTTCAAATCCAGG ATGGCCCAGCTGCGCCAGCATGC

(46) RFS sequences were amplified using the following PCR primers:

(47) TABLE-US-00005 AS764 SEQIDNO.14 TGATATCGGATGGCCCTGCTGCGCCAGGGAAAAGAAT AS845 SEQIDNO.15 GCCGCGGATTGCCCTTCTCCGACCCTGAC TobaccoRFSsequence SEQIDNO.16 GATATCGGATGGCCCTGCTGCGCCAGGGAAAAGAATAGAAGAAGCATCTG ACTACTTCATGCATGCTCCACTTGGCTCGGGGGGATATAGCTCAGTTGGT AGAGCTCCGCTCTTGCAATTGGGTCGTTGCGATTACGGGTTGGATGTCTA ATTGTCCAGGCGGTAATGATAGTATCTTGTACCTGAACCGGTGGCTCACT TTTTCTAAGTAATGGGGAAGAGGACCGAAACGTGCCACTGAAAGACTCTA CTGAGACAAAGATGGGCTGTCAAGAACGTAGAGGAGGTAGGATGGGCAGT TGGTCAGATCTAGTATGGATCGTACATGGACGGTAGTTGGAGTCGGCGGC TCTCCCAGGGTTCCCTCATCTGAGATCTCTGGGGAAGAGGATCAAGTTGG CCCTTGCGAACAGCTTGATGCACTATCTCCCTTCAACCCTTTGAGCGAAA TGCGGCAAAAGAAAAGGAAGGAAAATCCATGGACCGACCCCATCATCTCC ACCCCGTAGGAACTACGAGATCACCCCAAGGACGCCTTCGGCATCCAGGG GTCACGGACCGACCATAGAACCCTGTTCAATAAGTGGAACGCATTAGCTG TCCGCTCTCAGGTTGGGCAGTCAGGGTCGGAGAAGGGCAATCCGCGG ArabidopsisRFSsequence SEQIDNO.17 GATATCGGATGGCCCTGCTGCGCCAAGGAAAAGAATATAAGAAGGATCTG ACTCCTTCATGCATGCTCCACTTGGCTCGGGGGATATAGCTCAGTTGGTA GAGCTCCGCTCTTGCAATTGGGTCGTTGCGATTACGGGTTGGGTGTCTAA TTGTCCAGGCGGTAATGATAGTATCTTGTACCTGAACCGGTGGCTCACTT TTTCTAAGTAATGGGGAAAAGGACCGAAACATGCCACTGAAAGACTCTAC TGAGACAAAGATGGGCTGTCAAGAACGTAGAGGAGGTAGGATGGTCAGTT GGTCAGATCTAGTATGGATCGTACATGGACGGTAGTTGGAGTCGGCGGCT CTCCTAGGGTTCCCTCGTCTGGGATTGATCCCTGGGGAAGAGGATCAAGT TGGCCCTTGCGAACAGCTTGATGCACTATCTCCCTTCAACCCTTTGAGCG AAATGCGGCAAAAGGAAGGAAAATCCATGGACCGACCCCATCGTCTCCAC CCCGTAGGAACTACGAGATCACCCCAAGGACGCCTTCGGTATCCAGGGGT CGCGGACCGACCATAGAACCCTGTTCAATAAGTGGAATGCATTAGCTGTC CGCTCGCAGGTTGGGCAGTAAGGGTCGGAGAAGGGCAATCCGCGG

(48) Prrn promoter was amplified from tobacco genomic DNA cv. Petite Gerard using following PCR primers:

(49) TABLE-US-00006 AS750 SEQIDNO.18 GGCATGCCGCAATGTGAGTTTTTGTAGTTG Prrn-R SEQIDNO.19 ACTTGTATCGATGCGCTTCATATTCGCCCGGA Prrn16promotersequence SEQIDNO.20 GCATGCCGCAATGTGAGTTTTTGTAGTTGGATTTGCTCCCCCGCCGTCGT TCAATGAGAATGGATAAGAGGCTCGTGGGATTGACGTGAGGGGGCAGGGA TGGCTATATTTCTGGGAGCGAACTCCGGGCGAATATGAAGCGCATCGATA CAAGT

(50) aadA gene was synthesised by commercial DNA synthesis provider. Three introns from Arabidopsis gene At2g29890 were inserted into the coding sequence to optimise expression of the aadA in the cytoplasm of plant cells. The introns are underlined and shown in bold letters.

(51) TABLE-US-00007 aadAgenesequence SEQIDNO.21 ATGGCAGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAAG TAACTTTTAGCTCTCAGCTGCTGTTTACTAAGTTCATGCCATACATTGAT TCTGGTTTATTAAGGGTTATGTTCAGTATTACTAGTAACAAAATCTATTT CTTCGTTTCCGTCTGCAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACC GACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGA AGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGAT GAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTC CCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAGGTAATTTTCATCTTTG TTTGGCCTTCCAAGTGCTTTTTTTGCTGTTTACGGGTGGAACTTCAGTAA AAATGGGATCAAAACATCATATGGCATAAATAAATTTTAAGAATGGCGAA CTCGGGGTTACCGAATATGGCTTCCTTTTTCAGTGTTTCTTAGTCCATTG TACTTATGAGATTGCAGGTCACCATTGTTGTGCACGACGACATCATTCCG TGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAA TGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGG CTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCA GCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGC GCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCAGGTA AGAAATCTTTTCCCATCTTGAAGTCACCTCAAACCGAACGTTAGGAAATT CCAAAATGTTTTGATAGTAGTCTACTTAGTTTCAAGTTTTGGGTTTGTGT ATACTTTCACTAATAATATGCGTGGAAACATTGCAGGTGATGAGCGAAAT GTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAAT CGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCC AGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAA GAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTA CGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAA

(52) The psbA 3UTR terminator was amplified from the tobacco genomic DNA cv Petite Gerard using the following primers:

(53) TABLE-US-00008 AS749 SEQIDNO.22 GGATATCAAACAAATACAAAATCAAAATAGA AS778 SEQIDNO.23 GGAATTCTGAGCGCGCTAGAGCGATCCTG psbA3UTRsequence SEQIDNO.24 GAATTCTGAGCGCGCTAGAGCGATCCTGGCCTAGTCTATAGGAGGTTTTG AAAAGAAAGGAGCAATAATCATTTTCTTGTTCTATCAAGAGGGTGCTATT GCTCCTTTCTTTTTTTCTTTTTATTTATTTACTAGTATTTTACTTACATA GACTTTTTTGTTTACATTATAGAAAAAGAAGGAGAGGTTATTTTCTTGCA TTTATTCATGATTGAGTATTCTATTTTGATTTTGTATTTGTTTGATAT

(54) Primer Binding Domain (PBD) was designed as described by Friant et al., ((1998) Mol. Cellul. Biology, 18: 799-806) and amplified by PCR using the set of following overlapping primers:

(55) TABLE-US-00009 AS830 SEQIDNO.25 CCGCGGTATCTCACATTCACCCAATTGTCATGGTT AS831 SEQIDNO.26 TTAGAAGTATCCTGTGCACATCCGCAACCATGACAATTGG AS832 SEQIDNO.27 ACAGGATACTTCTAAGGAAGTCCACACAAATCAAGAACCCTTAGA AS833 SEQIDNO.28 TCACATTCTTCTGTTTTGGTAGCTGAAACGTCTAAGGGTTCTTGA AS834 SEQIDNO.29 CAGAAGAATGTGAGAAGGCTTCCACTAAGGCTAACTCTCAACAG AS835 SEQIDNO.30 CGCGGCCGCGTTGTCTGTTGAGAGTTAGC PBDsequence SEQIDNO.31 CCGCGGTATCTCACATTCACCCAATTGTCATGGTTGCGGATGTGCACAGG ATACTTCTAAGGAAGTCCACACAAATCAAGAACCCTTAGACGTTTCAGCT ACCAAAACAGAAGAATGTGAGAAGGCTTCCACTAAGGCTAACTCTCAACA GACAACGCGGCCGC

(56) LtrA gene from Lactococcus lactis encoded by the LtrB intron was synthesised by commercial DNA synthesis provider. The sequence of the LtrA protein was first optimised for codon usage in plants and 5 plant introns were inserted into the coding sequence to improve LtrA expression in plants. Plant introns inserted in the coding sequence of LtrA gene are underlined and shown in bold letters. The introns 1, 2 4 are from Arabidopsis gene At5g01290, intron 3 and 5 were selected from Arabidopsis gene At5g43940. The clone was named as LtrASi.

(57) TABLE-US-00010 LtrASigenesequence: SEQIDNO.32 GCATGCATGAAGCCAACAATGGCAATCCTCGAACGAATCTCTAAGAACTC ACAGGAGAACATCGACGAGGTACAATAACCCATATATATGAATTGATTCA TGTGTTACTCGTACTTGTTTGAATATGTTTGGAGCAAGTTTGATACTTTT GGATGATGATATCGCAAATTCGTTATCTTTTTGGCGTTATAGGTCTTCAC AAGACTTTACCGTTACCTTCTCCGTCCTGACATCTACTACGTGGCATATC AGAACCTCTACTCTAACAAGGGAGCTTCTACAAAGGGAATCCTCGATGAT ACAGCTGATGGATTCTCTGAGGAGAAGATCAAGAAGATCATCCAATCTTT GAAGGACGGAACTTACTACCCTCAGCCTGTCCGAAGAATGTACATCGCAA AGAAGAACTCTAAGAAGATGAGACCTCTTGGAATCCCAACTTTCACAGAC AAGTTGATCCAGGAGGCTGTGAGAATCATCCTTGAATCTATCTATGAGCC TGTCTTCGAGGATGTGTCTCACGGTTTCCGACCTCAGCGAAGCTGTCACA CAGCTTTGAAGACAATCAAGAGAGAGTTCGGAGGTAAATTATATGCTTTG CCACTTCCTCAAAAGATCATTTTAGGTTCATTGGTATGTGGTTTTTTTCT TAACAGGTGCAAGATGGTTCGTGGAGGGAGATATCAAGGGATGCTTCGAT AACATCGACCACGTCACACTCATCGGACTCATCAACCTTAAGATCAAGGA TATGAAGATGAGCCAGTTGATCTACAAGTTCCTCAAGGCAGGTTACCTCG AAAACTGGCAGTACCACAAGACTTACAGCGGAACACCTCAGGGCGGAATC CTCTCTCCTCTCCTCGCTAACATCTATCTTCATGAATTGGACAAGTTCGT TCTCCAACTCAAGATGAAGTTCGACCGAGAGAGTCCAGAGAGAATCACAC CTGAATACCGGGAGCTTCACAACGAGATCAAAAGAATCTCTCACCGTCTC AAGAAGTTGGAGGGCGAGGAGAAGGCTAAGGTTCTCTTGGAATACCAGGA GAAGAGGAAGAGGTTGCCTACACTCCCTTGTACATCACAAACAAACAAGG TTCGTTCTCTCCATTTTCATTCGTTTGAGTCTGATTTAGTGTTTTGTGGT TGATCTGAATCGATTTATTGTTGATTAGTGAATCAATTTGAGGCTGTGTC CTAATGTTTTGACTTTTGATTACAGGTCTTGAAGTACGTCCGATACGCTG ACGACTTCATCATCTCTGTTAAGGGAAGCAAGGAGGACTGTCAATGGATC AAGGAGCAATTGAAGCTCTTCATCCATAACAAGCTCAAGATGGAATTGAG TGAGGAGAAGACACTCATCACACATAGCAGTCAGCCTGCTCGTTTCCTCG GATACGACATCCGAGTCAGGAGAAGTGGAACTATCAAGCGATCTGGAAAG GTTCAATTCTTTCTTTCACATTTGTACTTGTTCACTCGTTTTATTAATCC TCTTTAGAATGGAGATTCTTACCTCTGTGTGGCCTTTGGCAGGTCAAGAA GAGAACACTCAACGGGAGTGTGGAGCTTCTCATCCCTCTCCAAGACAAGA TCCGTCAATTCATCTTCGACAAGAAGATCGCTATCCAGAAGAAGGATAGC TCATGGTTCCCAGTTCACAGGAAGTACCTTATCCGTTCAACAGACTTGGA GATCATCACAATCTACAACTCTGAATTGAGAGGTAAGCTGCTACCTCAAA CTTTCTAGTGCTTCCATATTTCCTTTCTTCTGCAAGGCAGAGAACCATTG TGGTTAAGTGTTTTAAATTGTGAATGTATAGGTATCTGCAACTACTACGG TCTCGCAAGTAACTTCAACCAGCTCAACTACTTCGCTTACCTTATGGAAT ACTCTTGCTTGAAGACTATCGCATCTAAGCATAAGGGAACACTCTCAAAG ACCATCTCTATGTTCAAGGATGGAAGTGGTTCTTGGGGAATCCCTTACGA GATCAAGCAGGGGAAGCAGAGGAGATACTTCGCCAACTTCAGTGAATGCA AATCTCCTTACCAATTCACTGATGAGATCAGTCAAGCTCCTGTGCTTTAC GGATACGCTCGGAACACTCTTGAGAACAGACTTAAGGCTAAGTGTTGTGA GCTTTGTGGAACATCTGATGAGAACACATCTTACGAGATCCACCACGTCA ACAAGGTCAAGAACCTTAAGGGAAAGGAGAAGTGGGAGATGGCAATGATC GCTAAGCAGCGGAAGACTCTTGTTGTTTGCTTCCATTGTCATCGTCACGT GATCCATAAGCACAAGTGAACTAGTAA

(58) The LtrA gene was translationally fused to the chloroplast transit peptide (rbcS-cTP) from tobacco Rubisco small subunit gene (Bank Access. No. AY220079) which was amplified using the following PCR primers:

(59) TABLE-US-00011 AS794 SEQIDNO.33 GCTCGAGACAATGGCTTCCTCAGTTCTTTCCTCT AS639 SEQIDNO.34 CGCATGCTACCTGCATACATTGCACTCTTCCACCAT rbcS-cTPsequence SEQIDNO.34 CTCGAGACAATGGCTTCCTCAGTTCTTTCCTCTGCAGCAGTTGCCACTCG CACCAATGTTGCTCAAGCTAACATGGTTGCACCTTTCACTGGTCTTAAGT CAGCTGCCTCATTCCCTGTTTCAAGGAAGCAAAACCTTGACATCACTTCC ATTGCTAGCAATGGTGGAAGAGTGCAATGTATGCAGGTAGCATGC

(60) The 5 promoter region from Arabidopsis ubiquitin 3 gene was amplifies with the following primers:

(61) TABLE-US-00012 AS724 SEQIDNO.35 CGGTACCTACCGGATTTGGAGCCAAGTC AS726 SEQIDNO.36 GTGTTTGGTGACCTGAAATAAAACAATAGAACAAGT ArabidopsisUbiq3promotersequence SEQIDNO.37 TACCGGATTTGGAGCCAAGTCTCATAAACGCCATTGTGGAAGAAAGTCTT GAGTTGGTGGTAATGTAACAGAGTAGTAAGAACAGAGAAGAGAGAGAGTG TGAGATACATGAATTGTCGGGCAACAAAAATCCTGAACATCTTATTTTAG CAAAGAGAAAGAGTTCCGAGTCTGTAGCAGAAGAGTGAGGAGAAATTTAA GCTCTTGGACTTGTGAATTGTTCCGCCTCTTGAATACTTCTTCAATCCTC ATATATTCTTCTTCTATGTTACCTGAAAACCGGCATTTAATCTCGCGGGT TTATTCCGGTTCAACATTTTTTTTGTTTTGAGTTATTATCTGGGCTTAAT AACGCAGGCCTGAAATAAATTCAAGGCCCAACTGTTTTTTTTTTTAAGAA GTTGCTGTTAAAAAAAAAAAAAGGGAATTAACAACAACAACAAAAAAAGA TAAAGAAAATAATAACAATTACTTTAATTGTAGACTAAAAAAACATAGAT TTTATCATGAAAAAAAGAGAAAAGAAATAAAAACTTGGATCAAAAAAAAA ACATACAGATCTTCTAATTATTAACTTTTCTTAAAAATTAGGTCCTTTTT CCCAACAATTAGGTTTAGAGTTTTGGAATTAAACCAAAAAGATTGTTCTA AAAAATACTCAAATTTGGTAGATAAGTTTCCTTATTTTAATTAGTCAATG GTAGATACTTTTTTTTCTTTTCTTTATTAGAGTAGATTAGAATCTTTTAT GCCAAGTATTGATAAATTAAATCAAGAAGATAAACTATCATAATCAACAT GAAATTAAAAGAAAAATCTCATATATAGTATTAGTATTCTCTATATATAT TATGATTGCTTATTCTTAATGGGTTGGGTTAACCAAGACATAGTCTTAAT GGAAAGAATCTTTTTTGAACTTTTTCCTTATTGATTAAATTCTTCTATAG AAAAGAAAGAAATTATTTGAGGAAAAGTATATACAAAAAGAAAAATAGAA AAATGTCAGTGAAGCAGATGTAATGGATGACCTAATCCAACCACCACCAT AGGATGTTTCTACTTGAGTCGGTCTTTTAAAAACGCACGGTGGAAAATAT GACACGTATCATATGATTCCTTCCTTTAGTTTCGTGATAATAATCCTCAA CTGATATCTTCCTTTTTTTGTTTTGGCTAAAGATATTTTATTCTCATTAA TAGAAAAGACGGTTTTGGGCTTTTGGTTTGCGATATAAAGAAGACCTTCG TGTGGAAGATAATAATTCATCCTTTCGTCTTTTTCTGACTCTTCAATCTC TCCCAAAGCCTAAAGCGATCTCTGCAAATCTCTCGCGACTCTCTCTTTCA AGGTATATTTTCTGATTCTTTTTGTTTTTGATTCGTATCTGATCTCCAAT TTTTGTTATGTGGATTATTGAATCTTTTGTATAAATTGCTTTTGACAATA TTGTTCGTTTCGTCAATCCAGCTTCTAAATTTTGTCCTGATTACTAAGAT ATCGATTCGTAGTGTTTACATCTGTGTAATTTCTTGCTTGATTGTGAAAT TAGGATTTTCAAGGACGATCTATTCAATTTTTGTGTTTTCTTTGTTCGAT TCTCTCTGTTTTAGGTTTCTTATGTTTAGATCCGTTTCTCTTTGGTGTTG TTTTGATTTCTCTTACGGCTTTTGATTTGGTATATGTTCGCTGATTGGTT TCTACTTGTTCTATTGTTTTATTTCAGGTCACCAAACA

(62) The nos terminator fragment was synthesised based on gene bank sequence accession EU048864.

(63) TABLE-US-00013 nosterminatorsequence SEQIDNO.38 TCTAGAGTCAAGCAGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGA TTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAA TTACGTGAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGA GATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATA GAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTC ATCTATGTTACTAGATCGACCTGCAG

(64) The reverse transcriptase-RNase H gene from yeast Ty1-H3 clone (Boeke et al., Mol. Cellul. Biology (1988), 8: 1432-1442; bank accession No. M18706) was optimised for codon usage in plants, and by insertion of 5 introns from Arabidopsis genome (intron 1from At1g04820, intron 2from At2g29550, intron 3from At1g31810, intron 4 and 5from At1g09170). The introns are underlined and shown in bold letters. The clone was synthesised by commercial DNA synthesis provider and named as RTRHi-Ty1.

(65) TABLE-US-00014 RTRHi-Ty1sequence SEQIDNO.39 ATGAACAATTCATCCCACAACATCGTTCCTATCAAGACTCCAACTACTGT TTCTGAGCAGAACACTGAAGAATCTATCATCGCTGATCTTCCACTTCCTG ATCTTCCTCCAGAATCTCCTACTGAATTTCCTGATCCATTCAAAGAACTT CCACCTATCAACTCAAGACAAACTAACTCTTCATTGGGCGGAATTGGCGA TTCTAATGCTTACACTACTATCAACTCTAAGAAGAGGTATTGTAGCCAGC CTCAACCAGTCTTTTTGCTGTTACATTTTCTTGGGCTCATCTAATGTTAT TTTCCTATTTTGTTTTCAGGTCACTTGAAGATAATGAAACTGAAATCAAA GTTTCTAGGGATACATGGAATACTAAGAATATGAGATCACTTGAACCTCC AAGATCTAAGAAGAGAATCCATCTTATTGCAGCTGTTAAAGCTGTGAAAT CAATCAAACCAATTAGAACAACTCTTAGATACGATGAAGCAATTACATAC AACAAAGACATCAAGGAGAAGGAGAAATACATCGAGGCTTACCACAAAGA AGTTAACCAACTTCTTAAGATGAAAACTTGGGATACTGATGAATACTACG ATAGAAAAGAGATTGACCCTAAGAGAGTTATCAACTCAATGTTCATCTTC AACAAGAAGAGAGACGGAACTCACAAAGCTAGATTCGTTGCAAGAGGAGA TATTCAGCATCCTGACACTTACGATTCAGGTAAGTATTCCAATGTTCTTC GATTATGAGTCAATGTTGTTACTGTATCTGTCTCTGTGGTTTATTGTTTC AGGCTTAGTTATTGATTAGTATTGAAACTTCACTCACATATTTTTTTGTT TGTTTTCTGAATTGTGCAGGTATGCAATCTAATACTGTTCATCACTACGC ATTGATGACATCTCTTTCACTTGCATTGGACAATAACTACTACATTACAC AACTTGACATATCTTCTGCATACCTTTACGCTGATATCAAGGAGGAGCTT TACATTAGACCTCCACCACATTTGGGAATGAATGATAAGTTGATCCGTTT GAAGAAATCACTTTACGGATTGAAACAATCTGGAGCTAATTGGTACGAAA CTATCAAATCATACCTTATTCAGCAATGCGGTATGGAGGAAGTTAGGGGA TGGTCATGCGTATTCAAGAACTCTCAAGTTACAATCTGCCTCTTCGTTGA TGATATGGTGCTCTTCTCTAAGAATCTTAACTCAAACAAGAGAATCATTG AGAAGTTGAAGATGCAATACGACACTAAGATCATCAACCTTGGAGAATCT GATGAGGAAATTCAATACGACATTCTTGGATTGGAAATCAAATACCAAAG AGGTGAGTTATATTTAACAGCTCATCAGTTACTTAAACACTTTTTGGGAC AAGCAGTTCAAACTCATGTTCCAATCCTAAAATTAATTGCAATTCACAGG TAAGTACATGAAGTTGGGAATGGAAAACTCATTGACTGAGAAGATTCCTA AACTTAACGTTCCTTTGAATCCAAAGGGAAGAAAGCTCTCTGCTCCAGGA CAACCAGGACTTTACATTGACCAGGATGAACTTGAGATTGATGAGGATGA ATACAAGGAGAAAGTACACGAGATGCAGAAGTTGATTGGACTTGCTTCAT ACGTTGGATACAAATTCAGATTCGACCTTCTTTACTACATCAACACACTT GCTCAGCATATACTTTTCCCATCTAGGCAAGTTCTTGACATGACATACGA GCTTATCCAATTCATGTGGGACACTAGAGACAAGCAACTCATATGGCACA AGAACAAGCCTACAGAGCCAGATAACAAGCTCGTTGCAATCTCTGATGCT TCTTACGGAAACCAACCATACTACAAATCACAAATTGGAAACATCTACTT GCTTAACGGAAAGGTACTTTTCTCAAAGACTTTACCTTATTGTGGAATAT TGAATTTTCTGAAAGACTTCACCTTATCTACATTTGTAATTTTACTATGG TAATCAGGTGATTGGAGGAAAGAGCACTAAGGCTTCACTTACATGCACTT CAACTACTGAGGCAGAGATCCACGCTATATCAGAATCTGTACCACTTCTT AACAACCTTTCTTACCTTATCCAAGAGCTTAACAAGAAGCCAATCATCAA GGGACTTCTTACTGACTCAAGATCAACAATCTCTATCATTAAGTCTACAA ATGAAGAGAAATTCAGAAACAGATTCTTCGGAACAAAGGCAATGAGACTT AGAGATGAAGTTTCAGGTAAGTATTAACTTACCAAATGATCAATATTATT TTGAAATGCAGGTTTTAGAATAATACTCTCTGCCGTTCTTGTTTATTTCC AGGTAACAACCTTTACGTTTACTACATCGAGACTAAGAAGAACATTGCTG ACGTTATGACAAAGCCTCTTCCTATCAAGACCTTCAAGTTGCTTACTAAC AAATGGATTCATTAA

(66) The RT-RH-Ty1 sequence was translationally fused to the chloroplast transit peptide from pea chloroplast HSP60 heat shock protein (Accession No. L03299). The sequence for the transit peptide (HSP60-cTP) was amplified from pea genomic DNA using the following PCR primers:

(67) TABLE-US-00015 AS293 SEQIDNO.40 TCTCGAGTTGATGGCTTCTTCTGCTCAAATA AS294 SEQIDNO.41 GGCATGCAACTCTCAAAGTGAAACCCTTC HSP60-cTPsequence SEQIDNO.42 CTCGAGATGGCTTCTTCTGCTCAAATACACGGTCTCGGAACCGCTTCTTT CTCTTCCCTCAAAAAACCCTCTTCCATTTCCGGTAATTCCAAAACCCTTT TCTTCGGTCAGCGACTCAATTCCAACCACTCTCCCTTCACCCGCGCCGCA TTCCCTAAGTTAAGTAGCAAAACCTTTAAGAAGGGTTTCACTTTGAGAGT TGCATGC

(68) The expression of the RTRHi-Ty1 and HSP60-cTP fusion was driven by TAF2 promoter from Arabidopsis taf2 gene. It was amplified from Arabidopsis genomic DNA (Col-0) using the following set of primers:

(69) TABLE-US-00016 AG3 SEQIDNO.43 GGTACCATGATCGCTTCATGTTTTTATC AG4 SEQIDNO.44 CTCGAGGTTCCTTTTTTGCCGATATGTTAG TAF2promotersequence SEQIDNO.45 GTACCATGATCGCTTCATGTTTTTATCTAATTTGTTAGCATATTGAATGA TTGATTTTCTTTTAATTTGGATATGTTGATTGTCTTGTTGCATCATCAAT GTATGTTTTATTTAACACCGGAAGATCTTATGATGGGTTCATTACTTCAT AATAATCTCCGAGTTCTACAAGACTACAACTTTCACGTGACTTTTACAGC GACAAAAAATGCATCTAGCGAAAATTAATCCACAACCTATGCATTTTTGT CACTCTTCACACGCGTATGTGCATAAATATATAGTATATACTCGACAATC GATGCGTATGTGTACACAATTACCAAAACAATTATTTGAATATTCAGACA TGGGTTGACATCACCAAGTAATATTCACAGTATCTGAAAACTATGTTTTG ACATCCCTAAATAGTTTGACTAACCAGTTTAATATGAGAGCATTTGTAAG AGGCAAGAGCCATGGTTTTGTTGGCTCGTTTAATATGCTCATTTAACCCC CCCAAAAAATACTATTAGATTTAAACGTAAAAGAATTAACGAACACAAGA ACTGCTAAAACAAAAAAAAATCAATGGCCGACATTTCATAGTTCATACAT CACTAATACTAAAAGATGCATCATTTCACTAGGGTCTCATGAAATAGGAG TTGACATTTTTTTTTGTAACGACAGAAGTTGACATGTTAAGCATCAATTT TTTTAAGAGTGGATTATACTAGTTTTTTTTTTTTTTTTTAATGTATGGTA TGATACAACAACAAAAACTATAAAATAGAAAAAGTCAGTGAAACCTCAAA TTGAAGGAAAAACTTTTGCACAAAAAGAGAGAGAGAGAGAAAGAATGTAA ATCCAAATAAATGGGCCTAATTGAGAATGCTTTAACTTTTTTTTTTTGGC TAAAAGAGAATGCTTTAACTAAGCCCATAAAATGAACATCAAACTCAAAG GGTAAGATTAATACATTTAGAAAACAATAGCCGAATATTTAATAAGTTTA AGACATAGAGGAGTTTTATGTAATTTAGGAACCGATCCATCGTTGGCTGT ATAAAAAGGTTACATCTCCGGCTAACATATCGGCAAAAAAGGAACCTCGA G

(70) The agropine synthase polyA signal (ags terminator) was synthesized based on the gene bank sequence EU181145.

(71) TABLE-US-00017 Theagsterminatorsequence SEQIDNo.46 GAATTAACAGAGGTGGATGGACAGACCCGTTCTTACACCGGACTGGGCGC GGGATAGGATATTCAGATTGGGATGGGATTGAGCTTAAAGCCGGCGCTGA GACCATGCTCAAGGTAGGCAATGTCCTCAGCGTCGAGCCCGGCATCTATG TCGAGGGCATTGGTGGAGCGCGCTTCGGGGATACCGTGCTTGTAACTGAG ACCGGATATGAGGCCCTCACTCCGCTTGATCTTGGCAAAGATATTTGACG CATTTATTAGTATGTGTTAATTTTCATTTGCAGTGCAGTATTTTCTATTC GATCTTTATGTAATTCGTTACAATTAATAAATATTCAAATCAGATTATTG ACTGTCATTTGTATCAAATCGTGTTTAATGGATATTTTTATTATAATATT GATGAT

(72) Plant Transformation

(73) Transformation of Arabidopsis Plants

(74) Transformation of Arabidopsis plants was performed as described by Clough & Bent (Clough & Bent (1998) Plant Journal 16:735-743). Agrobacterium tumefacience strain GV3101 (Koncz & Schell (1986) Mol Gen Genet 204:383-396) was used for transformation. Transformation of plants was carried out with chloroplast transformation constructs (FIG. 2) based on the pGreen 0029 binary vector (Hellens et al (2000) Plant Mol. Biol 42: 819-832). In brief, a chloroplast transformation cassette containing trnI flank, Prrn promoter, aadA gene, psbA 3 UTR, trnA flank and primer binding domain (PBD) was inserted into domain IV of the LtrB or fused to CTL from ASB using AscI-NotI enzymes. The resulting DNA fragment was fused to the 35S promoter and nos terminator and introduced into the pGreen0029 binary vector (EU048864). The fragment of LtrASi was fused to a chloroplast transit peptide (rbcS-cTP) and ubiq3 promoter from Arabidopsis. Resulting cassette was inserted into pGreen 0029 together with the chloroplast transformation cassette. The reverse transcriptase-RNase H (RTRHi-Ty1) was fused to HSP60-cTP transit peptide, TAF2 promoter and ags terminator. The resulted cassette was inserted in pSOUP vector (EU048870) carrying T-DNA from pGreen0179 vector (EU048866). The construct carrying the chloroplast cassette and LtrASi was co-transform with construct carrying RTRHi-Ty1 cassette in the same stain of Agrobacterium and used for Arabidopsis (Col-0) transformation.

(75) Transgenic lines were recovered on selection medium supplemented with 100 mg/l of spectinomycin.

(76) Transformation of Tobacco Plants

(77) Tobacco plants were transformed as described by Horsch et al., (1985) Science 227: 1229-1231, using Agrobacterium strain AGL1 (see protocol, below).

(78) The constructs were similar to the constructs used for Arabidopsis transformation with exception that trnI and trnA flanking sequences of the chloroplast cassette were amplified from tobacco genomic DNA (FIG. 2).

(79) Transgenic tobacco plants were regenerated on selection medium supplemented with 500 mg/l of spectinomycin.

(80) Transformation of Tobacco Leaf Explants with Agrobacterium Strain AGL1

(81) All items are autoclave-sterilised prior to use.

(82) Filter sterilize antibiotics to prevent fungal growth, keep antibiotics for plant tissue culture in separate box

(83) Sterilize plant material: take plants of about 9 cm high, they should not have started to flower. Cut leaves with cuticle (4-6 leaves per construct, enough to cut 100 explants), dip in 70% Ethanol and immediately dip in 1% Na-hypochlorite (cat. No 01032500; use bottle of bleach that is no more than 3 months old because the chlorine gas evaporates), hold leaves with forceps and stir in for 20 min. Avoid damaging the cuticle otherwise bleach will enter the vascular system. Rinse briefly in sterile water 5-6 times and leave in water until ready to be cut.

(84) Co-cultivation of agro with tobacco explants: grow AGL1 in LB or L broth with appropriate antibiotics overnight at 28-30 C., the next day re-suspend agro in co-cultivation solution so that the final concentration is around 0.4-0.6 OD.sub.600 nm. Place tobacco leaves in co-culture broth and cut squares of 1-1.5 cm1-1.5 cm with a rounded sterile scalpel using a rolling action. Dip the leaf explants in the agro solution with sterile forceps (stored in 100% ethanol, flamed and let to cool prior to touching the leaf tissue) blot on sterile Whatman paper and transfer on non-selective TSM plates (6 explants per plate) need to prepare about 15 plates per construct. Repeat this procedure for each construct, making sure that the scalpel and forceps, are dipped in ethanol and flamed between each construct to prevent cross-contamination. Leave for 2 days only for AGL1 (3-4 days for other agro strains)

(85) Transfer on selective TSM plates: use sterile flamed forceps to pick up and wash explants in 100 mls co-cultivation broth supplemented with timentin 320 mg/l (one pot per construct), shake well, blot on sterile whatman paper and place the washed explants on selective TSM plates supplemented with appropriate selective antibiotics and timentin 320 mg/l to kill agrobacterium.

(86) Shoot regeneration: takes around 1 month to see shoots appear, explants should be transferred on fresh plates every 10-14 days. Watch out for AGL1 recurrent growth, if Timentin is not enough to kill agro, add cefotaxime at 250 mg/l.

(87) Root regeneration: Takes around 1 week. Shoots are cut from the explants and place in growth boxes containing TRM supplemented with the appropriate selective antibiotics and timentin 320 mg/l+cefotaxime 250 mg/l to prevent agrobacterium recurrent growth.

(88) Maintain plants in TRM boxes: sub them every two weeks until ready to be transferred into glasshouse

(89) Adaptation to glasshouse conditions: soak peat pellets in sterile water until they swell to normal size and carefully place one plant per pellet, incubate the plants under 100% humidity conditions in a propagator, gradually opening the little windows until plants adapt to normal atmosphere over several days.

(90) Recipes:

(91) Co-culture: MS with vitamins and MES+0.1 mg/l NAA+1 mg/l BA+3% sucrose, pH 5.7

(92) TSM: MS with vitamins and MES+0.1 mg/l NAA+1 mg/l BA+3% sucrose, pH5.7, 0.2% gelrite

(93) TRM: MS salts with vitamins and MES+0.5% sucrose, pH5.7, 0.2% gelrite.

(94) Autoclave.

(95) Antibiotics Concentration

(96) For Agrobacterium LB or L Cultures:

(97) To grow AGL1 carrying pGreen/pSOUP: Carbenicillin 100 mg/l, Tetracycline 5 mg/ml, Rifampicin 50 mg/ml, Kanamycin 50 mg/ml

(98) AGL1 carrying pSOUP: Carbenicilin 100 mg/l, Tetracycline 5 mg/ml, Rifampicin 50 mg/ml.

(99) AGL1 empty: Carbenicillin 100 mg/l, Rifampicin 50 mg/ml.

(100) For Plant Culture:

(101) Kanamycin: 300 mg/l (100 mg/l if using benthamiana)

(102) Hygromycin: 30 mg/l (10 mg/l if using benthamiana)

(103) PPT: 20 mg/l (2 mg/l if using benthamiana)

(104) Spectinomycin: 500 mg/l

(105) Timentin: 320 mg/l. It is used to kill agro, fairly unstable make up small amount of stock, store in freezer for up to 1 month after that the antibiotic is no more efficient.

(106) Cefotaxime: 250 mg/l. Also used to kill agro, add to TS

(107) PCR Analysis of Transgenic Plants.

(108) The following primers have been used for amplification of flanking junction sequences:

(109) TABLE-US-00018 LFS1 SEQIDNO.47 GAGATGTGGATCATCCAAGGCA RFS1 SEQIDNO.48 CTACCATAGAGGCCAACGATAG AS527 SEQIDNO.49 AACGTCGGTTCGAGATGG(aadA-R1) aadA-F1 SEQIDNO.50 CGAAGGATGTCGCTGCCGACT;
and nested primers:

(110) TABLE-US-00019 SEQIDNO.51 LFS2 CTCCTCCTCAGGAGGATAGATG SEQIDNO.52 RFS2 AACTTTCATCGTACTGTGCTCTC SEQIDNO.53 AS526 GAGTCGATACTTCGGCGATC(aadA-R2) SEQIDNO.54 aadA-F2 CTAGACAGGCTTATCTTGGACA

(111) The following primers were used for amplification of chloroplast probe for Southern hybridisation:

(112) TABLE-US-00020 LP-F CGTGTTTAGTTGCCATCGTTGA SEQIDNO.55 LP-R GCTGAGAGCCCTCACAGCCCA SEQIDNO.56 RP-F TGTCAGCGGTTCGAGTCCGCTTA SEQIDNO.57 RP-R TAACCAAGCCACTGCCTATGAGT SEQIDNO.58

(113) The following primers were used for amplification of aadA gene as a probe for Northern hybridisation:

(114) TABLE-US-00021 aadA1 GTGATCGCCGAAGTATCGACT SEQIDNO.59 aadA2 ATCTCGCCTTTCACGTAGTGG SEQIDNO.60

(115) Results and Discussion

(116) The transformation of Arabidopsis and tobacco with our vectors containing transgene cassettes generated chloroplast transgenic plants by selection on medium supplemented with 100 mg/l of spectinomycin for Arabidopsis and 500 mg/l for tobacco (FIG. 2). In all cases we were able to detect insertion of the transgene cassette into the chloroplast genome using PCR amplification of junction regions. Five independent transgenic lines were analysed for all constructs and we could amplify correct size DNA fragment for insertion junctions in all lines. The amplified fragments were sequenced and correct insertion sites were confirmed.

(117) Southern and Northern analysis was also performed to confirm presence of insertion and the chloroplast transcripts.

EXPERIMENTAL SECTION 1B

(118) Modifications of the chloroplast transformation method used in Experimental section 1A can be improved using PBD designed for reverse transcription in the cytoplasm or in plastids, and by re-positioning of the building blocks on the transformation cassette (FIG. 3).

(119) The set of constructs was prepared for tobacco and rice transformation with LtrB intron (LtrB-CTS) or with ASB sequences (ASB-CTS) as the CTS (FIGS. 4-6). The positioning of transgene cassette building blocks was designed as described in FIG. 3, A-B for LtrB-CTS and FIG. 3, C-D for ASB-CTS.

(120) The PBD-CHL was designed as described previously.

(121) The primer binding domain of the tobacco tnt1 retrotransposon was used as the PBD-CYT, and it was amplified from genomic DNA of tobacco cv Petit Gerard using the following primers:

(122) TABLE-US-00022 AS912 SEQIDNO.61 GCCGCGGCTTTATTACCGTGAATATTA AS913 SEQIDNO.62 CGCGGCCGCTCTGATAAGTGCAACCTGATT PBD-CYT SEQIDNO.63 CTTTATTACCGTGAATATTATTTTGGTAAGGGGTTTATTCCCAACAACT GGTATCAGAGCACAGGTTCTGCTCGTTCACTGAAATACTATTCACTGTC GGTAGTACTATACTTGGTGAAAAATAAAAATGTCTGGAGTAAAGTACGA GGTAGCAAAATTCAATGGAGATAACGGTTTCTCAACATGGCAAAGAAGG ATGAGAGATCTGCTCATCCAACAAGGATTACACAAGGTTCTAGATGTTG ATTCCAAAAAGCCTGATACCATGAAAGCTGAGGATTGGGCTGACTTGGA TGAAAGAGCTGCTAGTGCAATCAGGTTGCACTTATCAGA

(123) In the first case, the PBD-CHL was fused to the 3 end of the LtrB intron (FIG. 3A, FIG. 4A for tobacco and FIG. 5A for rice). As LtrA protein possesses both LtrB-CTS-binding feature and reverse transcription activity it can fulfill both functions of the transgene RNA translocation into plastids and reverse transcription of the RNA cassette using plastid tRNA-Met as a primer.

(124) In the second case, the PBD-CYT was fused to CTU (FIG. 3B, FIG. 4B for tobacco and FIG. 5B for rice), so that reverse transcription of the transgene cassette is initiated and performed by endogenous reverse transcriptases in the cytoplasm using cytoplasmic tRNA-Met. The LtrA protein serves as CTS-binding peptide for translocation of RNA:DNA complex initiated by the reverse transcriptases into the plastids.

(125) The ASB-CTS was fused to the CTU with PBD-CHL or PDB-CYT (FIG. 3, C-D, FIGS. 6, A and B). The reverse transcriptase from yeast ty1 retrotransposon was co-delivered with construct containing PBD-CHL to facilitate reverse transcription reaction in the plastids.

(126) The chloroplast cassette for rice transformation was designed using rice-specific sequences (FIG. 5).

(127) The trnI fragment of the rice chloroplast genome was utilised as the LFS, and it was amplified using the following primers:

(128) TABLE-US-00023 AS699 SEQIDNO.64 GGCGCGCCGTGGGATCCGGGCGGTCCG AS700 SEQIDNO.65 GGCATGCTGGCGCAGCTGGGCCATCC RicetrnI-LFS SEQIDNO.66 Gggatccgggcggtccggggggggcactacggctcctctcttctcgagaa tccatacatcccttatcagtgtatggagagctatctctcgagcacaggtt gaggttcgtcctcaatgggaaaatggagcacctaacaacgcatcttcaca gaccaagaactacgagatcaccctttcattctggggtgacggagggatcg taccattcgagcctttttttcatgcttttcccggcggtctggagaaagca gcaatcaataggacttccctaatcctcccttcctgaaaggaagaacgtga aattctttttcctttccgcagggaccaggaggttggatctagccataaga ggaatgcttggtataaataagccacttcttggtcttcgactccctaagtc actacgagcgccctcgatcagtgcaatgggatgtggctatttatctatct cttgactcgaaatgggagcagagcaggtttgaaaaaggatcttagagtgt ctagggttgggccaggagggtctcttaacgccttcctttttctgcccatc ggagttatttcccaaggacttgccatggtaagggggagaaggggaagaag cacacttgaagagcgcagtacaacggagagttgtatgctgcgttcgggaa ggatgaatcgctcccgaaaaggagtctattgattctctcccaattggttg gatcgtaggggcgatgatttacttcacgggcgaggtctctggttcaagtc caggatggcccagctgcgcca

(129) The trnA fragment of the rice chloroplast genome was used as the RFS, and it was amplified using following primers:

(130) TABLE-US-00024 AS701 SEQIDNO.67 gatatcggatggcccagctgcgcca AS702 SEQIDNO.68 Gcggccgcattgcccttctccgaccct RicetrnA-RFS SEQIDNO.69 Ggatggcccagctgcgccagggaaaagaatagaagaagcatctgactctt tcatgcatactccacttggctcggggggatatagctcagttggtagagct ccgctcttgcaattgggtcgttgcgattacgggttggctgtctaattgtc caggcggtaatggtagtatcttgtacctgaaccggtggctcactttttct aagtaatggggaagaggactgaaacatgccactgaaagactctactgaga caaaaagatgggctgtcaaaaaggtagaggaggtaggatgggcagttggt cagatctagtatggatcgtacatggacgatagttggagtcggcggctctc ctaggcttccctcatctgggatccctggggaagaggatcaagttggccct tgcgaatagcttgatgcactatctcccttcaaccctttgagcgaaatgtg gcaaaaggaaggaaaatccatggaccgaccccattatctccaccccgtag gaactacgagatcaccccaaggacgccttcggcgtccaggggtcacggac cgaccatagaccctgttcaataagtggaacacattagccgtccgctctcc ggttgggcagtaagggtcggagaagggcaat

(131) The chloroplast-specific rrn16 promoter from wheat cv. Pavon was amplified using PCR with the following primers:

(132) TABLE-US-00025 AS518 SEQIDNO.70 TATCGATAACATTCCTCTAATTTCATTGCA AS720 SEQIDNO.71 GGCATGCAGGCTTGTGGGATTGACGTGATAG Wheatrrnpromotersequence(Wrrn) SEQIDNO.72 Aggcttgtgggattgacgtgatagggtagggttggctatactgctggtg gcgaactccaggctaataatctgaagcgcatggatacaagttatccttg gaaggaaagacaattccgaatctgctttgtctacgaataaggaagctat aagtaatgcaactatgaatctcatg aadA-mGFP4fusionsequence SEQIDNO.73 atggcagaagcggtgatcgccgaagtatcgactcaactatcagaggtag ttggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacattt gtacggctccgcagtggatggcggcctgaagccacacagtgatattgat ttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctt tgatcaacgaccttttggaaacttcggcttcccctggagagagcgagat tctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccg tggcgttatccagctaagcgcgaactgcaatttggagaatggcagcgca atgacattcttgcaggtatcttcgagccagccacgatcgacattgatct ggctatcttgctgacaaaagcaagagaacatagcgttgccttggtaggt ccagcggcggaggaactctttgatccggttcctgaacaggatctatttg aggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggc tggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagc gcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaa tggagcgcctgccggcccagtatcagcccgtcatacttgaagctagaca ggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcag ttggaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcg gcaaatcaggatccatgagtaaaggagaagaacttttcactggagttgt cccaattcttgttgaattagatggtgatgttaatgggcacaaattttct gtcagtggagagggtgaaggtgatgcaacatacggaaaacttaccctta aatttatttgcactactggaaaactacctgttccatggccaacacttgt cactactttctcttatggtgttcaatgcttttcaagatacccagatcat atgaagcggcacgacttcttcaagagcgccatgcctgagggatacgtgc aggagaggaccatcttcttcaaggacgacgggaactacaagacacgtgc tgaagtcaagtttgagggagacaccctcgtcaacaggatcgagcttaag ggaatcgatttcaaggaggacggaaacatcctcggccacaagttggaat acaactacaactcccacaacgtatacatcatggcagacaaacaaaagaa tggaatcaaagttaacttcaaaattagacacaacattgaagatggaagc gttcaactagcagaccattatcaacaaaatactccaattggcgatggcc ctgtccttttaccagacaaccattacctgtccacacaatctgccctttc gaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgta acagctgctgggattacacatggcatggatgaactatacaaataatcta ga

(133) atpA terminator was amplified from wheat DNA using the following primers:

(134) TABLE-US-00026 AS753 SEQIDNO.74 Accgcggtcaaataaattttgcatgtcta AS723 SEQIDNO.75 Gatatctccatactccttctttatgata WheatatpAterminator SEQIDNO.76 Caaataaattttgcatgtctactcttgttagtagaataggaatcgttga gaaagatttttcatttgaatcatgcaaaaaagttttctttgtttttagt ttagtatagttatttaaagaatagatagaaataagattgcgtccaatag gatttgaacctataccaaaggtttagaagacctctgtcctatccattag acaatggacgcttttctttcatattttattctttcttttattttttttt cttcttccgagaaaaaactgttagaccaaaactcttttaggaaatcaaa aaatccagatacaaatgcatgatgtatatattatatcatgcatatatca taaagaaggagtatgga

(135) The LtrA gene was driven by actin1 rice promoter amplified using the following primers:

(136) TABLE-US-00027 ARP1 SEQIDNO.77 gtcattcatatgcttgagaaga ARP2 SEQIDNO.78 gcctacaaaaaagctccgcacg Riceact1promotersequence SEQIDNO.79 gtcattcatatgcttgagaagagagtcgggatagtccaaaataaaacaa aggtaagattacctggtcaaaagtgaaaacatcagttaaaaggtggtat aagtaaaatatcggtaataaaaggtggcccaaagtgaaatttactcttt tctactattataaaaattgaggatgttttgtcggtactttgatacgtca tttttgtatgaattggtttttaagtttattcgcgatttggaaatgcata tctgtatttgagtcggtttttaagttcgttgcttttgtaaatacagagg gatttgtataagaaatatctttaaaaaacccatatgctaatttgacata atttttgagaaaaatatatattcaggcgaattccacaatgaacaataat aagattaaaatagcttgcccccgttgcagcgatgggtattttttctagt aaaataaaagataaacttagactcaaaacatttacaaaaacaaccccta aagtcctaaagcccaaagtgctatgcacgatccatagcaagcccagccc aacccaacccaacccaacccaccccagtgcagccaactggcaaatagtc tccacccccggcactatcaccgtgagttgtccgcaccaccgcacgtctc gcagccaaaaaaaaaaaaagaaagaaaaaaaagaaaaagaaaaacagca ggtgggtccgggtcgtgggggccggaaaagcgaggaggatcgcgagcag cgacgaggcccggccctccctccgcttccaaagaaacgccccccatcgc cactatatacatacccccccctctcctcccatccccccaaccctaccac caccaccaccaccacctcctcccccctcgctgccggacgacgagctcct cccccctccccctccgccgccgccggtaaccaccccgcccctctcctct ttctttctccgttttttttttcgtctcggtctcgatctttggccttggt agtttgggtgggcgagagcggcttcgtcgcccagatcggtgcgcgggag gggcgggatctcgcggctggcgtctccgggcgtgagtcggcccggatcc tcgcggggaatggggctctcggatgtagatctgcgatccgccgttgttg ggggagatgatggggggtttaaaatttccgccatgctaaacaagatcag gaagaggggaaaagggcactatggtttatatttttatatatttctgctg cttcgtcaggcttagatgtgctagatcttctttctttcttctttttgtg gtagaatttgaatccctcagcattgttcatcggtagtttttcttttcat gatttgtgacaaatgcagcctcgtgcggagcttttttgtaggc

(137) Transformation of Rice Immature Embryos.

(138) Immature Embryo Excision

(139) Day 1:

(140) Remove milky/post-milky stage immature seeds from panicles (immature embryos 1-2 mm in size are desired).

(141) Sterilize immature seeds: 50% sodium hypochlorite (12%)+1 drop of tween 20. Shake 10 min.

(142) Rinse 3-5 in sterile deionised, water. Drain off surplus water. Aliquot seeds (around 40) in sterile Petri dishes.

(143) Set up a 6015 mm Petri dish containing a 50% sodium hypochlorite solution and next to this a sterile beaker on its side with a sterile filter paper in it. Use sterile forceps to aseptically remove glumes from the first seed. Immerse this seed in the 50% sodium hypochlorite. Remove glumes from a second seed and immerse the second seed into the sodium hypochlorite solution whilst removing the first seed and storing this dehusked/sterilized seed on the filter paper in the beaker. Continue in this manner with all seeds.

(144) After all the glumes are removed:

(145) Sterilize dehusked seeds: 50% sodium hypochlorite: 5 min. with agitation.

(146) Rinse: 5-7 in sterile deionized water, drain.

(147) Place all seeds in a large sterile Petri dish. Aliquot for embryo excision (to keep seeds from drying out, work with only 50-100 in the plate at a time leaving the rest in the master plate).

(148) Remove the embryo from each seed and place embryo, scutellum up, in a 9015 mm Petri dish containing proliferation medium (40-50 embryos/plate). Culture at 28C in the dark for 2 days prior to bombardment

(149) Day 3:

(150) Check Each Embryo for Contamination Before Blasting

(151) Remove the embryos from the proliferation medium. Distribute 35-40 embryos scutellum upwards in an area 1 cm.sup.2 in the centre of a 6015 mm target plate containing 10 ml of proliferation medium+osmoticum (0.6M). Check each target plate so that the scutellum is straight. Allow enough room so the scutella do not shade each other out.

(152) Bombardment:

(153) TABLE-US-00028 Gun 14 kV Vacuum: 25 inches of Hg 1.sup.st bombardment 4 hours after osmoticum treatment 2.sup.nd bombardment 4 hours after 1.sup.st bombardment

(154) Day 4:

(155) 4-16 hours after the 2nd blast transfer immature embryos to proliferation medium without osmoticum. Culture in the dark at 28 C. for 2 days.

(156) Selection:

(157) Day 5:

(158) Aseptically cut out with scissors the germinating shoot. Transfer 16-20 immature embryos to fresh proliferation medium containing 30-50 mg/l Hygromycin (depending on the genotype); culture in the dark at 28 C.; record total number of embryos.

(159) After 10 days carefully remove the callus from the scutellum by breaking it up into 2-10 small pieces; subculture onto fresh proliferation medium+hygromycin. Do not subculture brown tissue and remaining immature embryo which could inhibit further growth of healthy callus.

(160) Subculture every 10 days by selecting healthy tissue: (embryogenic if present) and transfer it to fresh proliferation medium+hygromycin. Remove brown callus as it could be inhibiting to embryogenic callus.

(161) 30 to 40 days after bombardment change selection procedure. Instead of eliminating bad-looking tissue keep embryogenic tissue only (eliminate healthy non-embryogenic tissue)

(162) Regeneration:

(163) After 40 to 60 days, transfer established embryogenic callus showing differential growth on proliferation medium+hygromycin to regeneration medium+hygromycin. Culture at 28C under low light for 10 days then under high light for 10 additional days. Check plates periodically in the light for the development of embryos and green shoots. As shoots develop it is sometimes beneficial to gently move the developing shoot away from the callus it originated from and remove any dead tissue from the shoot itself to prevent inhibition of growth.

(164) Germination:

(165) Transfer white compact embryos and green shoots initiating roots to the germination medium under high light at 28C for 1 to 2 weeks. Check plates periodically. Remove necrotic tissue and divide germinating embryos if necessary.

(166) Results

(167) The analysis of transgenic plants was performed using PCR for insertion flanking sequences using the following primers for tobacco left flank:

(168) TABLE-US-00029 AS548 ACGGTGAAGTAAGACCAAGCTCAT SEQIDNO.80 AS549 CTAGGTCGGAACAAGTTGATAGGAC SEQIDNO.81
right flank:

(169) TABLE-US-00030 AS550 GGCTATGCCATCCTAAGGTGCTGCT SEQIDNO.82 AS551 CCATGAATGATAAATCATAGATCGAAC; SEQIDNO.83
for rice left flank:

(170) TABLE-US-00031 RC1 CCTGACCCGAAGATGTGGATC SEQIDNO.84 RC2 ACATTAGCATGGCGTACTCCT SEQIDNO.85
right flank

(171) TABLE-US-00032 RC3 AACCAGGAACGGGGAGCTCTC SEQIDNO.86 RC4 CGACTCTTTGATCTTAAACTT SEQIDNO.87

(172) Internal primers specific for aadA gene:

(173) TABLE-US-00033 AS526 GAGTCGATACTTCGGCGATC SEQIDNO.88 AS527 AACGTCGGTTCGAGATGG SEQIDNO.89
for mGFP gene

(174) TABLE-US-00034 AS528 TTACCAGACAACCATTACCTGTC SEQIDNO.90 AS529 GCTGGGATTACACATGGCAT SEQIDNO.91

(175) The expected size (1.1 kb) of PCR products were obtained for all tobacco constructs (FIG. 7). Sequencing analysis has confirmed junction site between transgene and plastid genome.

(176) Southern analysis has also confirmed transgene insertions into the correct location of the tobacco chloroplast genome (FIG. 8).

(177) Northern analysis indicated presence of transgene transcript in the fraction of the chloroplast RNA (FIG. 9).